The immune system is composed of many interdependent cell types that collectively protect the body from bacterial, parasitic, fungal, viral infections and from the growth of tumour cells. The guards of the immune system are macrophages that continually roam the bloodstream of their host. When challenged by infection or immunisation, macrophages respond by engulfing invaders marked with foreign molecules known as antigens. This event, mediated by helper T cells, sets forth a complicated chain of responses that result in the stimulation of B-cells. These B-cells, in turn, produce proteins called antibodies, which bind to the foreign invader. The binding event between antibody and antigen marks the foreign invader for destruction via phagocytosis or activation of the complement system. Five different classes of antibodies, or immunoglobulins, exist: IgA, IgD, IgE, IgG, and IgM. They differ not only in their physiological roles but also in their structures. From a structural point of view, IgG antibodies are a particular class of immunoglobulins that have been extensively studied, perhaps because of the dominant role they play in a mature immune response.
The biological activity, which the immunoglobulins possess, is today exploited in a range of different applications in the human and veterinary diagnostic, health care and therapeutic sector. In fact, in the last few years, monoclonal antibodies and recombinant antibody constructs have become the largest class of proteins currently investigated in clinical trials and receiving FDA approval as therapeutics and diagnostics. Complementary to expression systems and production strategies, purification protocols are designed to obtain highly pure antibodies in a simple and cost-efficient manner.
Traditional methods for isolation of immunoglobulins are based on selective reversible precipitation of the protein fraction comprising the immunoglobulins while leaving other groups of proteins in solution. Typical precipitation agents being ethanol, polyethylene glycol, lyotropic i.e. anti-chaotropic salts such as ammonium sulphate and potassium phosphate, and caprylic acid. Typically, these precipitation methods are giving very impure products while at the same time being time consuming and laborious. Furthermore, the addition of the precipitating agent to the raw material makes it difficult to use the supernatant for other purposes and creates a disposal problem, which is particularly relevant when speaking of large-scale purification of immunoglobulins.
Ion exchange chromatography is a well-known method of protein fractionation frequently used for isolation of immunoglobulins. However, since the charged ion exchange ligands will react with all oppositely charged compounds, the selectivity of ion exchange chromatography may be somewhat lower than other chromatographic separations.
Hydrophobic interaction chromatography (HIC) is another method described for isolation of immunoglobulins. However, hydrophobic matrices require an addition of lyotropic salts to the raw material to make the immunoglobulin bind efficiently. The bound antibody is released from the matrix by lowering the concentration of lyotropic salt in a continuous or stepwise gradient. If a highly pure product is the object, it is recommended to combine the hydrophobic chromatography with a further step. Thus, a disadvantage of this procedure is the necessity to add lyotropic salt to the raw material as this gives and problem and thereby increased cost to the large-scale user. For other raw materials than cell culture supernatants such as whey, plasma, and egg yolk the addition of lyotropic salts to the raw materials would in many instances be prohibitive in large-scale applications as the salt could prevent any economically feasible use of the immunoglobulin depleted raw material. An additional problem in large-scale applications would be the disposal of several thousand liters of waste.
Thiophilic adsorption chromatography was introduced by J. Porath in 1985 (J. Porath et al.; FEBS Letters, vol. 185, p. 306, 1985) as a new chromatographic adsorption principle for isolation of immunoglobulins. In this paper, it is described how divinyl sulphone activated agarose coupled with various ligands comprising a free mercapto-group show specific binding of immunoglobulins in the presence of 0.5 M potassium sulphate, i.e. a lyotropic salt. Although the matrices described for thiophilic chromatography generally show good performance, since lyotropic salts are added to the raw material to ensure efficient binding of the immunoglobulin, disadvantages as discussed above will arise.
Affinity chromatography occupies a unique and powerful role in separation technology as the only technique that enables purification of a biomolecule on the basis of biological function or individual chemical structure. High selectivity and high capacity make this technique ideally suited for the isolation of a specific substance from complex biological mixtures. In affinity chromatography, the molecule to be purified is specifically and reversibly adsorbed by a ligand comprising a complementary binding substance covalently attached to an insoluble support. The sample is applied under conditions which favour its specific binding to the immobilized ligand. Unbound substances are washed away and the substance of interest can be recovered by changing the experimental conditions to those which favour its desorption. Affinity chromatography has a concentrating effect, which enables convenient processing of large sample volumes. Protein A and Protein G affinity chromatography are popular and widespread methods for isolation and purification of immunoglobulins, particularly for isolation of monoclonal antibodies, mainly due to the ease of use and the high purity obtained.
In 1982, Colbert et al. described a gene coding for a protein A-like material. In U.S. Pat. No. 5,151,350, the successful cloning and expression of such genes was described for the first time. The cloning of this gene with its nucleotide sequence characterisation enables those skilled in the art to obtain quantities of a protein A-like material nucleotide sequence for cloning in various host-vector systems. Such recombinantly produced protein A-like material, and subfragments thereof, have the protein A properties of binding to IgG at the Fc region and activation of polyclonal antibody synthesis. Thus, these entities are useful in chromatography in the same manner as protein A. In the pharmaceutical industry, an obvious advantage of the recombinant protein A chromatography is that the risk of mammalian residues in the separation matrix, and consequently the risk of mammalian traces in the pharmaceutical product, has been eliminated.
U.S. Pat. No. 6,399,750 discloses an IgG-binding medium, and more specifically a separation medium having a base matrix and matrix-bound groups which exhibit recombinant Protein A (rProtein A) containing a cysteine. The groups are of formula: —B—X-rProtein A, wherein B is a bridge which binds to the base matrix and X includes a heteroatom N or S from rProtein. In a preferred embodiment X is a thioether sulphur originating which also constitutes the C-terminal residue is the cysteine of rProtein A.
Various Protein A chromatography products are available on the market. For example, Millipore (Billerca, Mass., USA) offers both Prosep-A High Capacity, made with natural protein A derived from Staphylococcus aureus and PROSEP-rA High Capacity, manufactured using recombinant protein A expressed in Escherichia coli. The PROSEP matrix consists of glass particles permeated by interconnecting pores.
McCue et al. (Journal of Chromatography A, 989 (2003) 139-153: “Evaluation of protein A chromatography media”) studied two protein A media of different pore sizes, both having porous glass backbones. A larger static capacity was found for the smaller pore size material, which is suggested to result from the larger specific surface area and associated higher ligand concentration. A larger dynamic binding capacity was also found for the smaller pore size material.
MabSelect™ is a Protein A chromatography product available from Amersham Biosciences (Uppsala, Sweden) especially suitable for capture of monoclonal antibodies from large volumes of feed. The ligands comprise recombinant Protein A coupled to a cross-linked agarose support via a C-terminal cysteine. The median particle diameter of MabSelect™ is 85 μm.
However, despite the state of the art constructions, there is still a need of alternative separation matrices for purification of antibodies or antibody constructs, which observe the demands of purity, safety, potency and cost effectiveness.